Project Summary RNA modifications (the epitranscriptome) represent a mechanism of post-transcriptional gene expression regulation and an emergent and exciting area of biology. N6-methyladenosine (m6A) is the most abundant post- transcriptional modification in mRNA, detected across the eukaryotic kingdom, from yeast to humans. m6A has been implicated in multiple molecular processes such as RNA splicing, RNA stability and miRNA processing and cellular functions such as meiosis, cell proliferation and embryonic stem cell differentiation, as well as disease states. The methyltransferases responsible for the m6A form a heterodimer in which METTL3 provides the catalytic activity. We and others have found that METTL3 has a positive impact in proliferation, differentiation and cell survival. Thus, we postulate that METTL3 represents a regulatory hub controlled by extra- and intracellular signals that allows the cells to respond to developmental cues and specific metabolic contexts by modulating RNA metabolism. Despite the rapid increase in our knowledge about the functions of m6A, there are many fundamental aspects of this process that remain unknown. For example, it is yet to be determined if the activity of the methyltransferases is regulated by upstream signaling pathways, and if this is the case, what are the mechanisms involved, and what are the molecular and cellular consequences of this regulation in homeostasis and development? Another key issue is to understand how the specificity of the methylation reaction is determined. We know that the recognition sequence for m6A methylation consists of a very short sequence motif ? but despite the abundance of this sequence in the transcriptome only a small fraction of such motifs gets methylated. Additional RNA structural motifs or sequence requirements have not been identified. To answer these broad questions, our lab is undertaking a multidisciplinary approach that includes complementary projects in the areas of molecular and cell biology, biochemistry as well as structural studies complemented with novel engineered mouse models. The program described here will allow us to solve the mechanisms involved in the regulation of the m6A methylation pathway upon extracellular stimulation, metabolic stress and development. Using state-of-the-art proteomic techniques, we will identify context-specific post- translational modifications acquired by METTL3, and their impact on the activity and specificity of the enzyme. We will complement these studies with structural techniques including X-ray crystallography and cryo-electron microscopy to define the molecular consequences of regulatory events on protein complex formation and substrate recognition. In parallel, we will use our recently developed mouse model that allows the inducible and tissue specific inactivation of METTL3 to distinguish between catalytic and non-catalytic functions of METTL3 and to understand the role of the m6A mark in stem cell self-renewal and differentiation.